Telomeres are specialized chromatin structures that protect the chromosome end and are comprised of short TG-rich sequence repeats and the proteins that bind them. Maintenance of the repeats requires telomerase, an enzyme-RNA complex whose internal RNA contains a template for the synthesis of new repeats, and in cells that have telomerase, telomere length is regulated such that the shortest telomeres in the cell are preferentially elongated. In the previous funding cycle, we showed that the cell cycle checkpoint kinase Tel1p is preferentially recruited to short telomeres and that its kinase activity is required for telomere elongation. We hypothesize that Tel1p recruitment to short telomeres stimulates elongation, at least in part, through phosphorylation of Rif1p, a component of telomere chromatin and known negative regulator of telomere length. Other work in the previous cycle revealed that the Tel1p paralog Mec1p has a distinct function from Tel1p, as it associates with telomeres when they have shortened to the point where they become dysfunctional and result in cell cycle arrest. We hypothesize that Mec1p association with dysfunctional telomeres stimulates elongation through phosphorylation of Tbf1p, a protein bound to the DNA adjacent to telomere repeats that has been implicated in the regulation of telomere elongation. In the previous cycle, we also developed toxic telomerase RNA alleles that greatly inhibit cell growth, and these alleles represent a potential therapy to treat cancer cells that express high levels of telomerase. Our long-term goals are to understand how telomeres are maintained and how they accomplish their unique cellular functions. We will pursue these goals 1) by testing the hypothesis that Tel1p telomere length control involves Rif1p phosphorylation (Aim 1), 2) by determining the processes that govern Mec1p recruitment to telomeres and that allow Mec1p-dependent telomere elongation (Aim 2), and 3) by defining how the toxic telomerase RNA alleles inhibit cell growth (Aim 3). Successful completion of these aims will make substantial contributions to our understanding of telomere length regulation in eukaryotes. PUBLIC HEALTH RELEVANCE: Telomeres are the physical ends of chromosomes and play important roles in chromosome maintenance and replication. Defects in telomere function have been implicated in cancer and a decreased life span of human stem cells, decreasing the body's capacity for repair. Using yeast as a model for human cells, we have identified key steps in telomere replication and telomere failure that we will investigate in this application. Understanding these mechanisms in yeast will provide a model for testing similar mechanisms in human cells.